The present invention relates to a novel sorbent comprising a spherical, highly porous polymer, and a process of solid phase extraction comprising the use of the novel sorbent.
Solid phase extraction is a sample preparation method that has seen rapid development during the last 15 years and has been established as a standard procedure in pharmaceutical and medical research, food chemistry, and environmental analytics. Solid phase extraction owes its rise to its versatility, economic efficiency, and easy automation. It is used for sample purification, sample concentration, solvent exchange (e.g. an analyt can be transferred from aqueous solution to an organic solution), desalinization, derivatization (an analyt is retained on the sorbent, derivatized, and then eluted), and for pre-separation e.g. into various substance classes.
The development of new combinatorial methods and fast analytical techniques such as liquid chromatography/mass spectrometry (LC-MS) has led to a significant acceleration in the development of new drugs. Regular creation of thousands of samples has driven the demand for methods that allow for high throughput in automated sample preparation in order to feed analytical methods and drug screening methods with purified samples. Solid phase extraction meets this challenge by providing powerful phases in 96-well and 384-well plate formats.
In modern trace analytics, sample preparation is often more important for the quality of the result than the instrumental analytical method used. This is especially true when analyzing traces in samples with complex compositions such as body fluids, biological tissues, food, extracts of natural products, and others. For such applications, solid phase extraction provides a time saving, reproducible alternative to otherwise used liquid-liquid extraction.
Solid phase extraction is an easily applied method using simple extraction cartridges of various dimensions, or high-throughput extraction plates in 96- or 384-well plate formats [Oasis Sample Extraction Products, Waters Inc., P/N 720000606EN, (2003)]. The basic principle of solid phase extraction is shown in FIG. 1. In a first step, the sorbent is typically conditioned. Then the usually aqueous sample solution is added to the top of the extraction cartridge (loading). While the sample solution flows through the sorbent bed, solutes are retained by the sorbent and thus extracted. Interferences (accompanying matrix substances) can then be removed selectively by choosing a suitable wash solution. Finally, the solute of interest is eluted selectively using a suitable solvent.
Alternatively to the process described above, the extraction cartridge can also be used to selectively retain sample impurities while the solute passes the sorbent unretained [Mitra, S. (editor), Sample preparation techniques in analytical chemistry, John Wiley & Sons (2003), p. 78-138].
In many applications solid phase extraction is used to concentrate the solute. To do so, a large volume of aqueous sample solution with low solute concentration is filtered through the sorbent and subsequently eluted with a small volume of organic solvent (usually methanol). In the ideal case, the complete amount of solute from the original aqueous solution will be contained in the methanol eluate.
Solid phase extraction cartridges are typically packed with sorbents having average particle sizes of 30 μm and 60 μm. Many solutions flow through the cartridge under the force of gravity. In order to treat solutions with higher viscosity one of the following methods is used:
vacuum at the outlet (lower end) of the cartridge
centrifugation of the sample through the cartridge
positive pressure at the inlet (upper end) of the cartridge
These methods are also recommended if the sorbent particle size is below 50 μm.
Most frequently a vacuum manifold is used to perform solid phase extraction. Positive pressure is only used when treating singular solutions, or in fully automated solid phase extraction systems. Manual addition of the sample using an injection syringe requires a cartridge adapter.
The most commonly used sorbents for solid phase extraction are based on so-called silica gel reverse phases (RP phases) whose polar surface has been modified with aliphatic, cycloaliphatic, and aromatic carbohydrates described in IE 0 059 565 B, EP 0 234 129 B1, and U.S. Pat. No. 4,680,121. These sorbents exhibit a number of disadvantages. They are unstable above pH=10 and below pH=2. They have to remain wetted during the full extraction procedure. Upon contact with purely aqueous solutions they exhibit a strong tendency to collapse. Such sorbents which have dried out or collapsed display poor retentions of analytes and therefore poor recovery.
The requirement that the sorbent remain wetted during the extraction process complicates the solid phase extraction process and significantly extends analysis time since instruments for automated solid phase extraction have to be equipped with additional sensors and safeguards to prevent drying out of the sorbent.
Copolymers of styrene and divinylbenzene are also known as polymer-based sorbents for solid phase extraction in addition to silica gel materials (U.S. Pat. No. 4,167,554, U.S. Pat. No. 4,495,250, U.S. Pat. No. 5,773,384). These polymer sorbents have strongly hydrophobic surface properties and display excellent pH resistance. However, similar to silica gel reversed phases, they must not dry out during the extraction process. If this requirement is not met, styrene-divinylbenzene-copolymers display poor recoveries for a number of substance classes.
Further progress in the field of polymer sorbents for solid phase extraction has been attained by U.S. Pat. No. 5,882,521 describing water-wettable polymer sorbents which have been introduced to the market in 1997. These sorbents are macroporous copolymers comprising two monomer components, the lipophilic divinylbenzene, and the hydrophilic N-vinylpyrrolidone, where the two monomers are present in a hydrophilic-hydrophobic balance (HLB). Because of its water-wettability this copolymer retains its capacity for strong retention of a broad spectrum of various analytes with good recoveries, even if the material dries out. In the dried out state, this copolymer shows much better retention (recovery greater than 85%) for polar analytes such as acetaminophen, ranitidine, or procainamide, than hydrophobic styrene-divinylbenzene-copolymers, and significantly better than classic silica gel C18 phases.
However, problems can arise with N-vinylpyrrolidone-divinylbenzene-copolymers when using strongly basic liquids as solvent, wash solution, or eluent. It is known that poly(N-vinylpyrrolidone) undergoes hydrolysis in the presence of bases to form poly(N-vinyl-aminobutyric acid) according to the following equation:
[Pielichowski J. J., Puszynski A. A., Technologia Tworzyw Sztucznych, Wydawnictwo WNT, Warszawa/Poland (1994)].
This reaction threatens the stability of the sorbent in basic environments. Moreover, the synthesis of N-vinylpyrrolidone-divinylbenzene-copolymers causes another problem since N-vinylpyrrolidone has been shown to be carcinogenic in animal tests [Römpp Chemie Lexikon, Thieme Verlag, Stuttgart N.Y., (1995)].
Thus, there is need for an alternative hydrophobic-hydrophilic polymer sorbent material which can be synthesized in an environmentally friendly way, exhibits high stability in basic and acidic media, and can be used as a universal solid phase extraction material in modern processes of solid phase extraction including high-throughput methods.